FLP-mediated gene modification in mammalian cells, and compositions and cells useful therefor

ABSTRACT

A gene activation/inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent β-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebriate development.

This application is a divisional application of U.S. Ser. No.08/147,912, filed Nov. 3, 1993, now pending, which is in turn acontinuation application of U.S. Ser. No. 07/666,252, filed Mar. 8,1991, now abandoned, the entire contents of each of which are herebyincorporated by reference herein.

This invention relates to recombinant DNA technology. In a particularaspect, this invention relates to methods for the site-specificrecombination of DNA in mammalian cells or host mammalian organisms. Inanother aspect, the present invention relates to novel DNA constructs,as well as compositions, cells and host organisms containing suchconstructs. In yet another aspect, the present invention relates tomethods for the activation and/or inactivation of expression offunctional genes. In a further aspect, the present invention relates tomethods for the introduction of DNA into specific sites in the genome ofmammalian cells. In a still further aspect, the present inventionrelates to gene therapy methods. In still another aspect, the presentinvention relates to means for the recovery of transfected DNA from acell or host organism. In a still further aspect, the present inventionrelates to assay methods.

BACKGROUND OF THE INVENTION

Many recent manipulations of gene expression involve the introduction oftransfected genes (transgenes) to confer some novel property upon, or toalter some intrinsic property of, mammalian cells or organisms. Theefficacy of such manipulations is often impaired by such problems as theinability to control the chromosomal site of transgene integration; orthe inability to control the number of copies of a transgene thatintegrate at the desired chromosomal site; or by difficulties incontrolling the level, temporal characteristics, or tissue distributionof transgene expression; or by the difficulty of modifying the structureof transgenes once they are integrated into mammalian chromosomes.

Transgenes are often introduced into mammalian cells or organisms todetermine which components of a transgene are required for specificqualitative or quantitative alterations of the host system. Since bothchromosomal position and copy number are major determinants of transgenefunction, the usefulness of these analyses is limited because currenttechniques for efficiently introducing transgenes into mammalian hostsresult in the insertion of a variable number of transgene copies atrandom chromosomal positions. It is, therefore, difficult (if notimpossible) to compare the effects of one transgene to those of anotherif the two transgenes occupy different chromosomal positions and arepresent in the genome at different copy numbers. Considerably morerefined analyses would be possible if one could routinely introducesingle copies of a variety of transgenes into a defined chromosomalposition.

The spatial or temporal characteristics of transgene expression isdifficult to control in intact organisms. The restricted expression oftransgenes is potentially of great interest, as this technique can beemployed for a variety of therapeutic applications, e.g., for theselective interruption of a defective gene, for the alteration ofexpression of a gene which is otherwise over-expressed orunder-expressed, for the selective introduction of a gene whose productis desirable in the host, for the selective removal or disruption of agene whose expression is no longer desired in the host, and the like.

Transgene expression is typically governed by a single set of controlsequences, including promoters and enhancers which are physically linkedto the transgenes (i.e., cis-acting sequences). Considerably greaterexpression control could be achieved if transgene expression could beplaced under the binary control of these cis-acting sequences, plus anadditional set of sequences that were not physically linked to thetransgenes (i.e., trans-acting sequences). A further advantage would berealized if the transient activity of these trans-acting functionsresulted in a stable alteration in transgene expression. In this manner,it would be possible, for example, to introduce into a host a transgenewhose expression would have lethal or deleterious effects if it wasconstitutively expressed in all cells. This would be accomplished bydelaying the expression of the transgene to a specific time ordevelopmental stage of interest, or by restricting the expression of thetransgene to a specific subset of the cell population.

It is currently difficult (if not impossible) to precisely modify thestructure of transgenes once they have been introduced into mammaliancells. In many applications of transgene technology, it would bedesirable to introduce the transgene in one form, and to then be able tomodify the transgene in a defined manner. By this means, transgenescould be activated or inactivated or the sequences which controltransgene expression could be altered by either removing sequencespresent in the original transgene or by inserting additional sequencesinto the transgene.

Previous descriptions of recombinase-mediated rearrangement ofchromosomal sequences in Drosophila and mammalian cells have notdirectly addressed the question of whether site-specific recombinasescould routinely create a functional translational reading frame.Moreover, the reported efficiency of the prior art recombinase system,in the only other description of site-specific recombination inmammalian cells reported to date based on Cre recombinase, described bySauer and Henderson in Nucleic Acids Research, Vol. 17: 147 (1989)!appears to be quite low.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have developed a system forthe selective modification of chromosomal or extrachromosomal DNA inmammalian cells. Selective modification can involve the insertion of oneDNA into another DNA (e.g., to create a hybrid gene, to activate a gene,to inactivate a gene, and the like), or the removal of specific DNAmolecule(s) from other DNA molecule(s) containing the DNA to be removed(e.g., to inactivate a gene, to bring desired DNA fragments intoassociation with one another, and the like).

The recombination system of the present invention is based onsite-specific recombinase, FLP. In one application of the inventionrecombination system, FLP-mediated removal of intervening sequences isrequired for the formation of a functional gene. Expression of thefunctional gene therefore, falls under the control of both theregulatory sequences associated with the functional gene and also underthe control of those sequences which direct FLP expression.

The reverse of the above-described process, i.e., the FLP-mediatedintroduction of DNA, provides a convenient and selective means tointroduce DNA into specific sites in mammalian chromosomes.

FLP-mediated recombination of marker genes provides a means to followthe fate of various sequences over the course of development and/or fromgeneration-to-generation. The recombination event creates a functionalmarker gene. This gain-of-function system can be used for lineageanalyses in a wide variety of tissues in different organisms. Prior toFLP-mediated recombination, the marker gene is normally silent, i.e.,the phenotype typical of the marker is not observed. In the absence ofFLP, spontaneous recombination to produce functional marker occurs onlyat very low frequencies. In the presence of FLP, functional marker isefficiently produced. In addition, this gain-of-function system isheritable and is easily detected by simple histochemical assays. Forexample, in transgenic mice, the lineages in which recombination is tooccur can be controlled by appropriate selection of the promoters usedto drive FLP expression. This could include promoters that are onlytransiently active at a developmental stage that substantially precedesovert cell differentiation. Since transcription of the marker gene iscontrolled by regulatory sequences associated therewith, functionalmarker genes can be expressed at later developmental stages, after celldifferentiation has occurred. By this means, it is possible to constructa map for mammalian development that correlates embryonic patterns ofgene expression with the organization of mature tissues.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. FIG. 1 presents schematic diagrams of FLP-mediatedrecombination events. In FIG. 1A, FLP-mediated introduction of DNA isillustrated, while in FIG. 1B, FLP-mediated removal of interveningsequences is illustrated.

FIGS. 2A, 2B, and 2C. FIG. 2 is presented in three parts. FIG. 2Apresents schematic diagrams of the expression vectors pFRTβGAL,pNEOβGAL, and pOG44 FLP. FIG. 2B presents a Southern blot of Hirtlysates prepared from 293 (human embryonic kidney) cells transfectedwith one microgram of pNEOβGAL and varying amounts of the pOG44 FLPexpression vector. FIG. 2C graphically presents the β-galactosidaseactivities in the same transfections shown in part B, referred to above.

FIGS. 3A and 3B. FIG. 3A, at the top, presents a schematic of thepattern of plasmid integration in E25 deduced from Southern blotanalysis. FIG. 3A, in the middle, presents the predicted pattern forβ-galactosidase positive subclones of E25 if precise recombinationacross the FLP-recombination target sites occurs. FIG. 3A, at thebottom, presents the predicted pattern for β-galactosidase negative,neomycin resistant subclones of E25B2 after FLP mediated insertion ofpOG45. FIG. 3B presents an analysis of genomic DNA from a cell line witha single integrated copy of pNEOβGAL (i.e., CVNEOβGAL/E25, designated asE25), two derivative β-galactosidase-positive subclones (designated asE25B1 and E25B2), and two subclones derived from E25B2 aftertransfection with pOG45 (designated as B2N1 and B2N2).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a mammalianrecombination system comprising:

(i) FLP recombinase, or a nucleotide sequence encoding same, and

(ii) a first DNA comprising a nucleotide sequence containing at leastone FLP recombination target site.

In accordance with another embodiment of the present invention, thereare provided novel DNA constructs useful for the introduction of DNAinto the genome of a transfected organism, said DNA constructcomprising, as an autonomous fragment:

(a) at least one FLP recombination target site,

(b) at least one restriction endonuclease recognition site,

(c) at least one marker gene,

(d) a bacterial origin of replication, and optionally

(e) a mammalian cellular or viral origin of DNA replication.

In accordance with yet another embodiment of the present invention,there are provided novel DNA constructs useful for the rescue of DNAfrom the genome of a transfected organism, said DNA constructcomprising, as an autonomous fragment, in the following order, readingfrom 5' to 3' along said fragment:

(a) a first FLP recombination target site,

(b) an insert portion comprising, in any suitable sequence:

(1) at least one restriction endonuclease recognition site,

(2) at least one marker gene,

(3) a bacterial origin of replication, and optionally

(4) a mammalian cellular or viral origin of DNA replication, and

(c) a second FLP recombination target site in tandem with said first FLPrecombination target site.

In addition, there are provided methods for the recovery of transfectedDNA from the genome of a transfected organism employing theabove-described constructs.

In accordance with still another embodiment of the present invention,there is provided a method for the assembly of a functional gene (whichis then suitable for activation of expression), in mammalian cells, byrecombination of individually inactive gene segments derived from one ormore gene(s) of interest, wherein each of said segments contains atleast one recombination target site, said method comprising: contactingsaid individually inactive gene segments with a FLP recombinase, underconditions suitable for recombination to occur, thereby providing a DNAsequence which encodes a functional gene of interest.

In accordance with a further embodiment of the present invention, thereis provided a method for the disruption of functional gene(s) ofinterest, thereby inactivating expression of such gene(s), in mammaliancells, wherein said gene(s) of interest contain at least one FLPrecombination target site, said method comprising contacting saidgene(s) of interest with:

(i) a DNA segment which contains at least one FLP recombination targetsite, and

(ii) FLP recombinase;

wherein said contacting is carried out under conditions suitable forrecombination to occur between said gene and said DNA segment, therebydisrupting the gene(s) of interest and rendering said gene(s)non-functional.

In accordance with a still further embodiment of the present invention,there is provided a method for the precisely targeted integration of DNAinto the genome of a host organism, said method comprising:

(i) introducing a FLP recombination target site into the genome of cellswhich are compatible with the cells of the subject,

(ii) introducing a first DNA comprising a nucleotide sequence containingat least one FLP recombination target site therein into the FLPrecombination target site in the genome of said cells by contacting saidcells with said first DNA and FLP recombinase, and thereafter

(iii) introducing the cells produced by the process of step (ii) intosaid subject, wherein the resulting cells and/or organism have theoptional ability to have DNA reproducibly and repetitively inserted intoand/or recovered from the host cells and/or organism.

In accordance with another aspect of the present invention, there areprovided mammalian cells, wherein the genomic DNA of said cells containat least one FLP recombination target site therein.

In accordance with yet another aspect of the present invention, thereare provided transgenic, non-human mammals, wherein said mammals containat least one FLP recombination target site in the genomic DNA thereof.

In accordance with yet another aspect of the present invention, there isprovided a method for the site-specific integration of transfected DNAinto the genome of the above-described cells and/or transgenic,non-human mammals, said method comprising:

(i) contacting said genome with:

(a) FLP recombinase, and

(b) a first DNA comprising a nucleotide sequence containing at least oneFLP recombination target site therein; and thereafter

(ii) maintaining the product of Step (i) under conditions suitable forsite-specific integration of said DNA sequence to occur at the FLPrecombination target site in said genome.

In accordance with a further aspect of the present invention, there isprovided a method for the analysis of the development of a mammal, saidmethod comprising:

(a) providing a transgenic mammal comprising:

(i) an expression construct encoding FLP under the control of aconditional promoter, and

(ii) a reporter construct under the control of the same or a differentpromoter, wherein said reporter construct encodes a functional ornon-functional reporter gene product, and wherein said constructcontains at least one FLP recombination target site therein,

wherein the functional expression of the functional reporter gene isdisrupted when said FLP recombination event occurs, or

wherein the functional expression of the non-functional reporter genecommences when said FLP recombination event occurs; and

(b) following the development of said mammal to determine whenexpression of functional reporter gene product either commences or isdisrupted.

In accordance with a still further aspect of the present invention,there is provided a co-transfection assay FLP-mediated recombination,said assay comprising:

(a) co-transfecting a host mammalian cell with:

(i) a FLP expression plasmid, and

(ii) a reporter plasmid comprising a reporter gene inactivated by thepresence of at least one recombination target site; and

(b) monitoring said host cell under a variety of conditions for the gainof expression of functional reporter gene product.

FLP recombinase is a protein which catalyzes a site-specificrecombination reaction that is involved in amplifying the copy number ofthe 2μ plasmid of S. cerevisiae during DNA replication. FLP protein hasbeen cloned and expressed in E. coli see, for example, Cox, inproceedings of the National Academy of Sciences U.S.A., Vol. 80:4223-4227 (1983)!, and has been purified to near homogeneity see, forexample, Meyer-Lean, et al., in Nucleic Acids Research, Vol. 15:6469-6488 (1987)!. FLP recombinases contemplated for use in the practiceof the present invention are derived from species of the genusSaccharomyces. Preferred recombinases employed in the practice of thepresent invention are derived from strains of Saccharomyces cerevisiae.Especially preferred recombinases employed in the practice of thepresent invention are proteins having substantially the same amino acidsequence as set forth in Sequence I.D. No. 2, as encoded, for example,by Sequence I.D. No. 1, or the sequence set forth by Hartley andDonelson, Nature 286: 860 (1980).

The FLP recombination target site (sometimes referred to herein as"FRT") has also been identified as minimally comprising two 13 base-pairrepeats, separated by an 8 base-pair spacer, as follows: ##STR1## Thenucleotides in the above "spacer" region can be replaced with any othercombination of nucleotides, so long as the two 13 base-pair repeats areseparated by 8 nucleotides. The actual nucleotide sequence of the spaceris not critical, although those of skill in the art recognize that, forsome applications, it is desirable for the spacer to be asymmetric,while for other applications, a symmetrical spacer can be employed.Generally, the spacers of the FLP recombination target sites undergoingrecombination with one another will be the same.

As schematically illustrated in FIG. 1A, contact of genomic DNAcontaining a FLP recombination target site (shown as the linearPsv-BETA-GAL construct) with a vector containing a FLP recombinationtarget site, in the presence of the protein, FLP recombinase, results inrecombination that forms a new genomic sequence wherein the vectorsequences have been precisely incorporated into the genome of the host.The reverse of this process is shown schematically in FIG. 1B, wherein agenomic sequence or construct containing two tandemly oriented FLPrecombination target sites, upon contacting with FLP, is recombined andthe FLP recombination target site-bounded fragment is excised as acircular molecule.

Genes of interest contemplated for use in the practice of the presentinvention can be selected from genes which provide a readily analyzablefunctional feature to the host cell and/or organism, e.g., visiblemarkers (such as β-galactosidase, thymidine kinase, tyrosinase, and thelike), selectable markers, (such as markers useful for positive andnegative selection, e.g., genes for antibiotic resistance), as well asother functions which alter the phenotype of the recipient cells, andthe like.

The first DNA employed in the practice of the present invention cancomprise any nucleotide sequence containing at least one FLPrecombination target site, which will precisely define the locus atwhich FLP-mediated recombination will occur. The nucleotide sequence cancomprise all or part of a gene of interest, as well as other sequencesnot necessarily associated with any known gene. Optionally, for ease oflater recovery of the gene of interest (in "activated" or modifiedform), the first DNA can optionally contain a second FLP recombinationtarget site.

The second DNA employed in the practice of the present invention isselected from at least a second portion of the first gene of interest orat least a portion of a second gene of interest (including an intactform of a second gene of interest). When the second DNA is at least asecond portion of the first gene of interest, the site-specificrecombination of the present invention may act to provide a functionalcombination of the different portions of the first gene of interest.Alternatively, when the second DNA is at least a portion of a secondgene of interest, the site-specific recombination of the presentinvention may act to provide a functional hybrid gene, which produces aproduct which is not identical with either the product of the first geneor the second gene. As yet another alternative, when the second DNA is aportion of a second gene, the site-specific recombination of the presentinvention may act to disrupt the function of the first gene of interest.Based on the nature of the first DNA and the second DNA, as well as themode of interaction between the two, the site-specific interaction ofthe present invention may create or disrupt a feature which iscolorimetrically detectable, immunologically detectable, geneticallydetectable, and the like.

In accordance with the present invention, assembly of a functionalexpression unit is achieved in any of a variety of ways, e.g., byassociation of the gene of interest with a functional promoter, byassembly of common gene fragments to produce a complete functional gene(which, in combination with its promoter, comprises a functionalexpression unit), or assembly of diverse gene fragments from diversesources to produce a functional, hybrid gene (which, in combination witha promoter, comprises a functional expression unit), and the like. Uponassembly of a functional expression unit as described herein, expressionof the functional gene to produce a protein product can be activated inthe usual manner. In the absence of FLP-mediated recombination,activation of expression would fail to produce a functional proteinproduct.

In accordance with the present invention, dis-assembly of a functionalexpression unit is achieved in any of a variety of ways, e.g., bydis-associating the gene of interest from a functional promoter, bydis-assembly (e.g., disruption) of the functional gene (e.g., byintroduction of DNA which renders the entire sequence non-functional),by removal of a substantial portion of the coding region of said gene,and the like. Upon dis-assembly of a functional expression unit asdescribed herein, expression of the functional gene product under theconditions required prior to gene dis-assembly is no longer possible.The ability of the expression unit to be activated for expression hastherefore been disrupted. The gene in this situation can be said to beinactivated, since activation of expression is not possible.

Individually inactive gene segments contemplated for use in the practiceof the present invention are fragments which, alone, do not encodefunctional products. Such fragments can be derived from a first gene ofinterest alone, or from both a first and second gene of interest DNAfragments.

When gene inactivation is desired, the gene of interest can be disruptedwith a DNA fragment which throws the gene of interest out of readingframe (e.g., an insert wherein the number of nucleotides is not amultiple of 3). Alternatively, the gene of interest can be disruptedwith a fragment which encodes a segment which is substantiallydissimilar with the gene of interest so as to render the resultingproduct non-functional. As yet another alternative, the gene of interestcan be disrupted so as to dis-associate the gene of interest from thetranscriptional control of the promoter with which it is normallyassociated.

The introduction of DNA, e.g., DNA encoding FLP recombination targetsites, into the genome of target cells can be accomplished employingstandard techniques, e.g., transfection, microinjection,electroporation, infection with retroviral vectors, and the like.

Introduction of protein, e.g., FLP recombinase protein, to host cellsand/or organisms can be accomplished by standard techniques, such as forexample, injection or microinjection, transfection with nucleotidesequences encoding FLP, and the like.

When employed for gene therapy of an intact organism, introduction oftransgenic cells into the subject is accomplished by standardtechniques, such as for example, grafting, implantation, and the like.

Mammalian cells contemplated for use in the practice of the presentinvention include all members of the order Mammalia, such as, forexample, human cells, mouse cells, rat cells, monkey cells, hamstercells, and the like.

Host organisms contemplated for use in the practice of the presentinvention include each of the organism types mentioned above, with theproviso, however, that no claim is made to genetically modified humanhosts (although the present invention contemplates methods for thetreatment of humans).

Once FLP recombinase (or DNA encoding same) and DNA containing at leastone FLP recombination target site have been introduced into suitablehost cells/organisms, the cells/host organisms are maintained underconditions suitable for the site-specific recombination of DNA. Suchconditions generally involve conditions required for the viability ofthe host cell or organism. For in vitro manipulations, conditionsemployed typically involve low concentrations of a variety of buffershaving a pH of between about 5-9 and ionic strengths in the range ofabout 50-350 mM. See, for example, Senecoff, et al., in Journal ofMolecular Biology, Vol. 201: 405-421 (1988).

In accordance with a particular aspect of the present invention, aco-transfection assay has been developed which can be used tocharacterize FLP-mediated recombination of extrachromosomal DNA in avariety of cell lines. Cells are co-transfected with an expressionconstruct and a "reporter" plasmid that is a substrate for therecombinase. The expression construct encodes a FLP recombinase protein.The reporter plasmid encodes either a functional reporter genecontaining at least one recombination target site therein, or anon-functional reporter gene containing at least one recombinationtarget site therein. Upon expression of FLP by the expression construct,the functional reporter gene will be rendered non-functional, or thenon-functional reporter gene will be rendered functional. Thus, theactivity of the expression construct can be assayed either by recoveringthe reporter plasmid and looking for evidence of recombination at theDNA level, or by preparing cytoplasmic extracts and looking for evidenceof recombination at the protein level (i.e., by measuring the expressionof reporter gene activity generated by the recombined reporter). Suchassays are described in greater detail in Example 1 below.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES Example 1

Co-transfection Assays.

The co-transfection assay used to characterize FLP-mediatedrecombination of extrachromosomal DNA involved transfection of cellswith an expression construct and a "reporter" plasmid that was asubstrate for the recombinase. The activity of the expression constructcould be assayed either by recovering the reporter plasmid and lookingfor molecular evidence of recombination at the DNA level, or bypreparing cytoplasmic extracts and looking for evidence of recombinationat the protein level (i.e., by measuring β-galactosidase activitygenerated by recombined reporter).

The pNEOβGAL reporter plasmid used for these assays was derived frompFRTβGAL (FIG. 2A). In the Figure, half-arrows indicate positions of FLPrecombination target (FRT) sites; E and S designate EcoRI and ScaIrestriction sites, respectively; Psv designates early promoter fromSV40; BETA-GAL designates the β-galactosidase structural sequence; NEOdesignates neomycin expression cassette; Pcmv designates thecytomegalovirus immediate early promoter; IN designates an intron; FLPdesignates a FLP coding sequence; AN designates an SV40 adenylationcassette; thin lines represent vector sequences; and the sizes ofrestriction fragments are indicated in kb.

pFRTβGAL contains a version of the bacterial β-galactosidase sequencemodified by insertion of a FLP recombination target site, or FRT, withinthe protein coding sequence immediately 3' to the translational start.The oligonucleotide used for the construction of pFTβRGAL was: ##STR2##This oligonucleotide contains an in-frame start codon, minimal FRT site,and an additional copy of the 13-bp FRT repeat °XXX°!; the XbaI sitewithin the FRT spacer is enclosed in parentheses. The linker wasinserted between the BamHI and HindIII sites of pSKS105 (M. J.Casadaban, A. Martin-Arias, S. K. Shapira, and J. Chou, Meth Enzymol.100, 293 (1983)) and the LacZ portion of modified gene was cloned into apSV2 vector. The neomycin cassette used for construction of pNEOβGAL wasan XhoI to BamHI fragment from pMClneo-polyA (K. Thomas and M. Capecchi,Cell 51: 503 (1987)) cloned between copies of the J3 FRT site in pUC19.

The FRT consists of two inverted 13-base-pair (bp) repeats and an 8-bpspacer that together comprise the minimal FRT site, plus an additional13-bp repeat which may augment reactivity of the minimal substrate. Theβ-galactosidase translational reading frame was preserved upon insertionof the FRT site, and the resulting plasmid, pFRTβGAL, generated robustactivity in mammalian cells (Table 1).

pNEOβGAL was constructed by cutting pFRTβGAL in the middle of the FRTsite with XbaI and then inserting an XbaI fragment consisting of twohalf-FRT sites flanking a neomycin transcription unit. This createdintact FRTs on either side of the neomycin cassette and rendered theβ-galactosidase transcription unit inactive (Table 1). PreciseFLP-mediated recombination of the FRTs caused the excission of theneomycin cassette, recreated the parental pFRTβGAL plasmid, and restoredβ-galactosidase expression.

Co-transfection of cells with a fixed amount of pNEOβGAL reporterplasmid and increasing amounts of the pOG44 FLP expression vectorgenerated increasing amounts of recombined reporter plasmid andconsequently, increased levels of β-galactosidase activity. Molecularevidence for FLP-mediated recombination was obtained by recoveringplasmids 36 hours after transfection, followed by endonuclease treatment(with EcoRI and ScaI) and Southern blotting (see FIG. 2B; employing as aprobe the fragment of pFRTβGAL indicated at the top of FIG. 2A). Lysatesof cells from cotransfections that included the pOG44 FLP expressionvector showed a signal at 5.6 kb, the position at which recombinedreporter (equivalent to pFRTβGAL) would run, and a 3.2 kb signal thatwas generated by unrecombined pNEOβGAL reporter (FIG. 2A). The 5.6 kbband intensity was proportional to the amount of FLP expression plasmidincluded in the transfection. The 5.6 kb band was not seen incotransfections in which a non-FLP plasmid was substituted for the FLPexpression vector (FIG. 2B) or in transfections that contained onlypOG44 (and no reporter plasmid). pOG44 generated additional signals at2.2 kb and 2.8 kb because the plasmid used in its construction containedEcoRI and EcoRI-ScaI fragments of such length.

pOG44 consists of the cytomegalovirus immediate early promoter frompCDM8 see Aruffo and Seed in Proc. Natl Acad. Sci. , USA 84: 8573(1987)!, a 5' leader sequence and synthetic intron from pMLSIScat seeHuang and Gorman in Nucl. Acids Res. 18: 937 (1990)!, the FLP codingsequence (bp 5568-6318 and 1-626 of the 2 μm circle, see Hartley andDonelson, Nature 286: 860 (1980)! and the SV40 late regionpolyadenylation signal from pMLSIScat. The following silent nucleotidesubstitutions were introduced into the structural FLP sequence using thepolymerase chain reaction: C for T at position 5791, G for A at 5794, Gfor C at 5800, C for T at 55, G for A at 58, and C for T at 103. Thesechanges eliminated three cannonical AATAAA polyadenylation signals andintroduced a PstI restriction site without altering the amino acidsequence encoded by the nucleotide sequence. pOG28 consists of a murinecDNA for dihydrofolate reductase cloned into pCDM8 (Aruffo and Seed,supra).

In the same samples, β-galactosidase activity was also proportional tothe amount of FLP expression plasmid included (FIG. 2C). Only backgroundactivities were observed in cotransfections that included a non-FLPcontrol plasmid (Table 1) or when pOG44 alone was transfected. Theexperiment thus provides both molecular and biochemical evidence forprecise FLP-mediated recombination in mammalian cells.

Table 1 presents β-galactosidase activities in cotransfection assays of293, CV-1, and F-9 cells. Positive control transfections (pFRTβGAL)included 1 μg of pFRTβGAL and 18 μg of the pOG28 non-FLP controlplasmid; negative control transfections (pNEOβGAL) included 1 μg ofpNEOβGAL and 18 μg of the pOG28; and experimental transfections(pNEOβGAL+FLP) contained 1 μg of pNEOβGAL and 18 μg of the pOG44 FLPexpression plasmid (FIG. 1A). The column headed by "%" shows thepNEOβGAL+FLP values as a percentage of the pFRTβGAL positive control.Each value represents the mean for six plates from two experiments.Standard errors are in parentheses. Neither pOG28 nor pOG44 generatedβ-galactosidase activity when transfected alone. All transfectionscontained 1 μg of pRSVL de Wet et al., Mol. Cell. Biol. 7: 725 (1987)!to correct β-galactosidase activities for relative transfectionefficiencies.

Subconfluent cultures of cells in 10 cm dishes and grown in Dulbecco'smodified Eagle's medium (DMEM) and 5% calf serum were transfected byovernight exposure to calcium phosphate precipitates Graham et al.,Virology 36: 59 (1979)! and then split 1:4. After 24 hours incubation,one plate of each transfection was harvested by Hirt extraction J. MolBiol. 26: 365 (1967)! and a second plate was used to prepare cytoplasmicextracts de Wet et al., supra!. Approximately 5% of the DNA recoveredfrom single plates was used for Southern analyses. β-galactosidaseassays were performed as described by Hall et al., in J. Mol. Appl.Genet. 2: 101 (1983)!. Luciferase activities generated by the inclusionof 1 μg of pRSVL (de Wet et al., supra) in all transfections were usedto correct β-galactosidase activities for relative transfectionefficiencies. The experiment was repeated twice with similar results.

                  TABLE 1                                                         ______________________________________                                        β-GALACTOSIDASE ACTIVITIES (UNITS/MG PROTEIN)                            IN COTRANSFECTED CELLS                                                        TRANSFECTIONS                                                                                               pNEOβGAL +                                 CELL LINE                                                                             pFRTβGAL                                                                            pNEOβGAL                                                                            FLP      %                                      ______________________________________                                        293     30.4 (1.9) 0.17 (0.02)                                                                              14.2 (2.2)                                                                             47                                     CV-1    275 (25)   0.33 (0.06)                                                                              22.6 (1.2)                                                                             8                                      F9      24.8 (4.3) 0.04 (0.01)                                                                              1.88 (0.02)                                                                            8                                      ______________________________________                                    

FLP activity has also been demonstrated in monkey kidney (CV-1) andmouse embryonal carcinoma (F9) cells. In Table 1, the β-galactosidaseactivity in the "pFRTβGAL" transfections represents an estimate of theexpression expected if all the pNEOβGAL in a co-transfection wereimmediately recombined. The highest β-galactosidase expression inco-transfections employing pNEOβGAL plus pOG44, relative to pFRTβGALtransfected cells, was 47%, seen in 293 cells. This is a remarkablelevel considering that β-galactosidase expression required both FLPexpression, followed by recombination of pNEOβGAL, to produce aconstruct capable of expressing β-galactosidase. Co-transfections ofCV-1 and F9 cells generated 8% of the activity seen in the pFRTβGALtransfections. Even at this lower relative activity, cotransfected cellswere readily observed in histochemical reactions for β-galactosidaseactivity.

Example 2

FLP-Mediated Removal of Intervening Sequences

If the invention method is to be widely applicable, for example for geneactivation in transgenic mammals, the ability of FLP to faithfullypromote precise recombination at FLP recombination target sitescontained in the mammalian genome is required. Such ability isdemonstrated in this example.

Cell lines that contain single integrated copies of pNEOβGAL (designatedCVNEOβGAL/E) were produced by transfecting CV-1 cells with linearizedplasmid by electroporation, then isolated by selecting G418-resistant(G418^(R)) transfectants that stably expressed the neomycin cassette,and finally identifying single copy lines by Southern blot analyses(FIG. 3). As previously shown for other integrated constructs withsimilarly short direct repeats, the chromosomal FRTs did notspontaneously recombine (in the absence of FLP) to produce aβ-galactosidase-positive (βGAL⁺) phenotype at detectable frequencies(Table 2).

Transient expression of FLP in the CVNEOβGAL/E lines (by transientlytransfecting with the pOG44 FLP expression vector) promoted a rapidconversion to a βGAL⁺ phenotype. When five different lines weretransiently transfected with the pOG44 FLP expression vector,β-galactosidase activities at 36 hours were 40 to 100-fold higher thanthose seen in replicate plates transfected with a non-FLP plasmid (Table2). At 48 hours after transfection histochemical processing showed manypositive cells (Table 2).

Table 2 presents the β-galactosidase phenotypes of CVNEOβGAL/E lines,which contain a single copy of the β-galactosidase inactive reporter,pNEOβGAL, after transfection with FLP expression (pOG44), non-FLPnegative control (pOG28) or β-galactosidase positive control (pFRTβGAL)plasmids. The pFRTβGAL transfections included 1 μg of pFRTβGAL and 19 μgof pOG44; other mixes contained 20 μg of the indicated plasmid.β-galactosidase activities are mean values for triplicate transfectionsperformed as described for FIG. 2 and assayed 36 hours after removal ofprecipitates; standard errors for the pOG44 transfections were less than10% of the mean. The percent positive was determined by scoring morethan 10³ cells after transfection and histochemical processing asdescribed by de Wet et al., supra.

                  TABLE 2                                                         ______________________________________                                        β-GALACTOSIDASE PHENOTYPES OF                                            TRANSFECTED CVNEOβGAL CELL LINES                                                ACTIVITIES                                                             CELL   (units/mg protein)                                                                         PERCENT POSITIVE                                          LINE  pOG28     pOG44   pOG28   pFRTβGAL                                                                         pOG44                                 ______________________________________                                        E6    0.24      11.2    0†                                                                              8.7     6.1                                  E25   0.21      16.7    0†                                                                             17.1    12.4                                  E26   0.18      7.2     0†                                                                             19.5    15.4                                  E14   0.28      13.1    ND      ND      ND                                    E22   0.09      9.6     ND      ND      ND                                    ______________________________________                                         †No positive cells were found among >10.sup.6 cells examined.          ND: Not done.                                                            

To provide some estimate of the efficiency of recombination, anadditional set of replicate plates were transfected with the pFRTβGALβ-galactosidase expression vector. Comparing the fractions of cells thatwere βGAL+ in the pFRTβGAL and in the pOG44 transfections (assumingsimilar transfection efficiencies) suggests that most (70-80%) of thecells transfected with pOG44 were converted to a βGAL⁺ phenotype (Table2). The comparison undoubtedly underestimates the efficiency ofFLP-mediated excision. Whereas many copies of a functionalβ-galactosidase gene were available for immediate transcription in thepositive controls, recombination may have occurred shortly beforeharvest in some pOG44-transfected cells. In these cases the singlerecombined reporter gene may not have generated enough β-galactosidaseby the time of harvest to render the cells positive in this assay.

The βGAL⁺ phenotype was passed on to all descendents of manyFLP-converted cells. Positive colonies were formed during prolongedexpansion of individual colonies. Entirely negative colonies and mixedcolonies were also observed. Mixed colonies would be expected ifrecombination occurred after mitosis in only one descendent of atransfected cell, or if recombined and unrecombined cells mixed atreplating or during subsequent growth. Indeed, the physical segregationof phenotypes evident in most mixed colonies suggested that they werecomposed of stably positive and negative lineages.

The correlation between β-galactosidase expression and recombination atFRT sites was examined by comparing the structure of the integratedpNEOβGAL sequences in two βGAL⁺ subclones to the parental line.CVNEOβGAL/E25 (106) cells were transfected with the pOG44 FLP expressionvector and subcloned 12 hours after removal of the precipitate. Afterhistochemical screening, two βGAL⁺ subclones (E25B1 and E25B2) wereexpanded for further analysis. In Southern blots of genomic DNA fromboth subclones, the pattern of hybridization matched that expected forFLP-mediated recombination of the FRT sites in the parental line (FIG.3). While recombination products have not been recovered and sequenced,these Southern analyses and the fact that activation of β-galactosidaseexpression required creation of a functional translational reading frameindicate that FLP-mediated recombination was precise.

Example 3

FLP Mediated Recombination of FRT on an Extrachromosomal Molecule With aChromosomally Integrated FRT.

Reversal of the process described in the previous Example, i.e., theFLP-mediated recombination of an FRT site on a plasmid with achromosomally integrated FRT site, can be used to target the integrationof transfected plasmids to specific genomic sites. To determine thefrequency at which this occurs, G418-sensitive, βGAL⁺ E25B2 cells wereco-transfected with the pOG44 FLP expression vector and a plasmid,pOG45, that contained a neomycin resistance gene expression cassette anda single FRT. pOG45 consisted of the neomycin resistance cassette and 3'FRT from pNEOβGAL cloned into pUC19.8 8×10⁵ CVNEOβGAL cells weretransfected by electroporation in 800 μl of saline containing 40 μg ofpOG44 and 0.1 μg of either pOG45 or, for a negative control, pOG45A(which was derived from pOG45 by deleting a 200 bp fragment containingthe FRT).

G418^(R) subclones (designated B2N) from three transfections that hadstably integrated pOG45 were histochemically stained for β-galactosidaseactivity and more than half (104 of 158, or ⁻ 66%) were either entirelyβ-galactosidase-negative (βGAL⁻) or predominantly βGAL⁻ with a fewclusters of βGAL⁺ cells. The remaining colonies were βGAL⁺. Withcontinued passage as dispersed monolayers, the fraction of βGAL⁺ cellsin the mosaic lines rapidly diminished. This suggested they were G418⁻sensitive cells that initially survived because of their proximity toresistant cells; this was confirmed by reconstitution experiments. Allof the 55 colonies formed after parallel co-transfections of pOG44 and aderivative of pOG45 (pOG45A) that lacked an FRT were βGAL⁺.

The correlation between loss of β-galactosidase activity andrecombination between plasmid and chromosomal FRTs was examined inSouthern analyses. Because the FRT and neomycin cassette of pOG45 werederived from the neomycin cassette and 3' FRT of pNEOβGAL (FIG. 2A),recombination of the plasmid FRT with the E25B2 chromosomal FRTregenerates the 3.2 kb EcoRI fragment of the original CVNEOβGAL/E25parent. Additionally, the 8.5 kb junctional fragment of CVNEOβGAL/E25shifts to 12.0 kb because pOG45 is 3.5 kb larger than the neomycincassette of pNEOβGAL. The 3.2 kb EcoRI fragment and the 8.5 kbjunctional fragment were observed in each of the 10 cell lines analyzedafter initial histochemical classification as βGAL⁻ or mosaic, as shownfor two such lines in FIG. 3B. In contrast, each of the four βGAL+colonies examined by Southern analyses showed that pOG45 had integratedat a random site.

These data show that FLP-mediated recombination will target theintegration of transfected DNA to a specific chromosomal site atfrequencies that exceed those of random integration, and that the eventcan be marked by the alteration in gene activity at the target site. Theefficiency of targeted integration can be increased by standardoptimization techniques, such as for example, by using ratios of theintegrating plasmid and FLP expression vectors different from the singleratio mixture used here, or by using FRT mutations in the plasmid andchromosomal sites to decrease the frequency with which successfullyintegrated plasmids are subsequently excised.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

SUMMARY OF SEQUENCES

Sequence I.D. No. 1 is the approximately 1450 base-pair sequenceencoding a FLP recombinase contemplated for use in the practice of thepresent invention, as well as the amino acid sequence deduced therefrom.

Sequence I.D. No. 2 is the amino acid sequence deduced from thenucleotide sequence of Sequence ID No. 1.

Sequence I.D. No. 3 is the nucleotide sequence of the FLP recombinationtarget site (FRT).

Sequence I.D. No. 4 is the nucleotide sequence of the oligonucleotideused in the construction of pFTβRGAL.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1380 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: NATIVE FLP                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1269                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGCCACAATTTGATATATTATGTAAAACACCACCTAAGGTGCTTGTT48                            MetProGlnPheAspIleLeuCysLysThrProProLysValLeuVal                              151015                                                                        CGTCAGTTTGTGGAAAGGTTTGAAAGACCTTCAGGTGAGAAAATAGCA96                            ArgGlnPheValGluArgPheGluArgProSerGlyGluLysIleAla                              202530                                                                        TTATGTGCTGCTGAACTAACCTATTTATGTTGGATGATTACACATAAC144                           LeuCysAlaAlaGluLeuThrTyrLeuCysTrpMetIleThrHisAsn                              354045                                                                        GGAACAGCAATCAAGAGAGCCACATTCATGAGCTATAATACTATCATA192                           GlyThrAlaIleLysArgAlaThrPheMetSerTyrAsnThrIleIle                              505560                                                                        AGCAATTCGCTGAGTTTCGATATTGTCAATAAATCACTCCAGTTTAAA240                           SerAsnSerLeuSerPheAspIleValAsnLysSerLeuGlnPheLys                              65707580                                                                      TACAAGACGCAAAAAGCAACAATTCTGGAAGCCTCATTAAAGAAATTG288                           TyrLysThrGlnLysAlaThrIleLeuGluAlaSerLeuLysLysLeu                              859095                                                                        ATTCCTGCTTGGGAATTTACAATTATTCCTTACTATGGACAAAAACAT336                           IleProAlaTrpGluPheThrIleIleProTyrTyrGlyGlnLysHis                              100105110                                                                     CAATCTGATATCACTGATATTGTAAGTAGTTTGCAATTACAGTTCGAA384                           GlnSerAspIleThrAspIleValSerSerLeuGlnLeuGlnPheGlu                              115120125                                                                     TCATCGGAAGAAGCAGATAAGGGAAATAGCCACAGTAAAAAAATGCTT432                           SerSerGluGluAlaAspLysGlyAsnSerHisSerLysLysMetLeu                              130135140                                                                     AAAGCACTTCTAAGTGAGGGTGAAAGCATCTGGGAGATCACTGAGAAA480                           LysAlaLeuLeuSerGluGlyGluSerIleTrpGluIleThrGluLys                              145150155160                                                                  ATACTAAATTCGTTTGAGTATACTTCGAGATTTACAAAAACAAAAACT528                           IleLeuAsnSerPheGluTyrThrSerArgPheThrLysThrLysThr                              165170175                                                                     TTATACCAATTCCTCTTCCTAGCTACTTTCATCAATTGTGGAAGATTC576                           LeuTyrGlnPheLeuPheLeuAlaThrPheIleAsnCysGlyArgPhe                              180185190                                                                     AGCGATATTAAGAACGTTGATCCGAAATCATTTAAATTAGTCCAAAAT624                           SerAspIleLysAsnValAspProLysSerPheLysLeuValGlnAsn                              195200205                                                                     AAGTATCTGGGAGTAATAATCCAGTGTTTAGTGACAGAGACAAAGACA672                           LysTyrLeuGlyValIleIleGlnCysLeuValThrGluThrLysThr                              210215220                                                                     AGCGTTAGTAGGCACATATACTTCTTTAGCGCAAGGGGTAGGATCGAT720                           SerValSerArgHisIleTyrPhePheSerAlaArgGlyArgIleAsp                              225230235240                                                                  CCACTTGTATATTTGGATGAATTTTTGAGGAATTCTGAACCAGTCCTA768                           ProLeuValTyrLeuAspGluPheLeuArgAsnSerGluProValLeu                              245250255                                                                     AAACGAGTAAATAGGACCGGCAATTCTTCAAGCAATAAACAGGAATAC816                           LysArgValAsnArgThrGlyAsnSerSerSerAsnLysGlnGluTyr                              260265270                                                                     CAATTATTAAAAGATAACTTAGTCAGATCGTACAATAAAGCTTTGAAG864                           GlnLeuLeuLysAspAsnLeuValArgSerTyrAsnLysAlaLeuLys                              275280285                                                                     AAAAATGCGCCTTATTCAATCTTTGCTATAAAAAATGGCCCAAAATCT912                           LysAsnAlaProTyrSerIlePheAlaIleLysAsnGlyProLysSer                              290295300                                                                     CACATTGGAAGACATTTGATGACCTCATTTCTTTCAATGAAGGGCCTA960                           HisIleGlyArgHisLeuMetThrSerPheLeuSerMetLysGlyLeu                              305310315320                                                                  ACGGAGTTGACTAATGTTGTGGGAAATTGGAGCGATAAGCGTGCTTCT1008                          ThrGluLeuThrAsnValValGlyAsnTrpSerAspLysArgAlaSer                              325330335                                                                     GCCGTGGCCAGGACAACGTATACTCATCAGATAACAGCAATACCTGAT1056                          AlaValAlaArgThrThrTyrThrHisGlnIleThrAlaIleProAsp                              340345350                                                                     CACTACTTCGCACTAGTTTCTCGGTACTATGCATATGATCCAATATCA1104                          HisTyrPheAlaLeuValSerArgTyrTyrAlaTyrAspProIleSer                              355360365                                                                     AAGGAAATGATAGCATTGAAGGATGAGACTAATCCAATTGAGGAGTGG1152                          LysGluMetIleAlaLeuLysAspGluThrAsnProIleGluGluTrp                              370375380                                                                     CAGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGAAGCATACGATAC1200                          GlnHisIleGluGlnLeuLysGlySerAlaGluGlySerIleArgTyr                              385390395400                                                                  CCCGCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCA1248                          ProAlaTrpAsnGlyIleIleSerGlnGluValLeuAspTyrLeuSer                              405410415                                                                     TCCTACATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGCACT1299                       SerTyrIleAsnArgArgIle                                                         420                                                                           ATGCCGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCGACGTGA1359              ACAGTGAGCTGTATGTGCGCA1380                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 423 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetProGlnPheAspIleLeuCysLysThrProProLysValLeuVal                              151015                                                                        ArgGlnPheValGluArgPheGluArgProSerGlyGluLysIleAla                              202530                                                                        LeuCysAlaAlaGluLeuThrTyrLeuCysTrpMetIleThrHisAsn                              354045                                                                        GlyThrAlaIleLysArgAlaThrPheMetSerTyrAsnThrIleIle                              505560                                                                        SerAsnSerLeuSerPheAspIleValAsnLysSerLeuGlnPheLys                              65707580                                                                      TyrLysThrGlnLysAlaThrIleLeuGluAlaSerLeuLysLysLeu                              859095                                                                        IleProAlaTrpGluPheThrIleIleProTyrTyrGlyGlnLysHis                              100105110                                                                     GlnSerAspIleThrAspIleValSerSerLeuGlnLeuGlnPheGlu                              115120125                                                                     SerSerGluGluAlaAspLysGlyAsnSerHisSerLysLysMetLeu                              130135140                                                                     LysAlaLeuLeuSerGluGlyGluSerIleTrpGluIleThrGluLys                              145150155160                                                                  IleLeuAsnSerPheGluTyrThrSerArgPheThrLysThrLysThr                              165170175                                                                     LeuTyrGlnPheLeuPheLeuAlaThrPheIleAsnCysGlyArgPhe                              180185190                                                                     SerAspIleLysAsnValAspProLysSerPheLysLeuValGlnAsn                              195200205                                                                     LysTyrLeuGlyValIleIleGlnCysLeuValThrGluThrLysThr                              210215220                                                                     SerValSerArgHisIleTyrPhePheSerAlaArgGlyArgIleAsp                              225230235240                                                                  ProLeuValTyrLeuAspGluPheLeuArgAsnSerGluProValLeu                              245250255                                                                     LysArgValAsnArgThrGlyAsnSerSerSerAsnLysGlnGluTyr                              260265270                                                                     GlnLeuLeuLysAspAsnLeuValArgSerTyrAsnLysAlaLeuLys                              275280285                                                                     LysAsnAlaProTyrSerIlePheAlaIleLysAsnGlyProLysSer                              290295300                                                                     HisIleGlyArgHisLeuMetThrSerPheLeuSerMetLysGlyLeu                              305310315320                                                                  ThrGluLeuThrAsnValValGlyAsnTrpSerAspLysArgAlaSer                              325330335                                                                     AlaValAlaArgThrThrTyrThrHisGlnIleThrAlaIleProAsp                              340345350                                                                     HisTyrPheAlaLeuValSerArgTyrTyrAlaTyrAspProIleSer                              355360365                                                                     LysGluMetIleAlaLeuLysAspGluThrAsnProIleGluGluTrp                              370375380                                                                     GlnHisIleGluGlnLeuLysGlySerAlaGluGlySerIleArgTyr                              385390395400                                                                  ProAlaTrpAsnGlyIleIleSerGlnGluValLeuAspTyrLeuSer                              405410415                                                                     SerTyrIleAsnArgArgIle                                                         420                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: FLP recombination target site                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC34                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (C) INDIVIDUAL ISOLATE: Synthetic oligonucleotide                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATCCCGGGCTACCATGGAGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAG60                GAACTTCA68                                                                    __________________________________________________________________________

That which is claimed is:
 1. A composition that effects recombination inmammalian cells comprising:(i) an isolated and purified FLP recombinase,or an isolated and purified nucleotide sequence encoding same, and (ii)a first DNA comprising a nucleotide sequence containing a first FLPrecombination target site (FRT) therein,wherein the genome of themammalian cells contains a stably integrated second FRT site, andwherein said FLP recombinase catalyzes recombination between said firstFRT and the second FRT, thereby precisely targeting integration of saidfirst DNA into the genome.
 2. A composition according to claim 1,wherein said first DNA comprises at least a first segment of a firstgent of interest, said composition further comprising:(iii) a second DNAcomprising:(a) at least a second segment of said first gene of interest,or (b) at least a segment of a second gent of interest; wherein saidsecond DNA contains at least one FLP recombination target site; andwherein said second DNA, when combined in reading frame with said firstDNA, provides a functional gene.
 3. A composition according to claim 2wherein said second DNA comprises said second segment of said first geneof interest.
 4. A composition according to claim 2 wherein said secondDNA comprises said segment of said second gene of interest.
 5. Acomposition according to claim 4 wherein said segment of said secondgene of interest, when combined in reading frame with said first DNA,provides a hybrid, functional gene.
 6. A composition according to claim4 wherein said segment of said second gene of interest, when combinedwith said first DNA, disrupts the function of said first gene ofinterest.
 7. A composition according to claim 1 wherein said first DNAfurther comprises a third FRT.
 8. A composition according to claim 1wherein said FLP recombinase is from a species of the genusSaccharomyces.
 9. A composition according to claim 1 wherein said FLPrecombinase is from a strain of Saccharomyces cerevisiae.
 10. Acomposition according to claim 9 wherein said FLP recombinase is encodedby the sequence set forth as SEQ ID NO:1.
 11. A composition according toclaim 1 wherein said first DNA provides a readily analyzable marker uponexpression.
 12. A composition according to claim 2 wherein said secondDNA provides a readily analyzable marker upon expression.
 13. Acomposition according to claim 1 wherein said first DNA is an autonomousfragment comprising:(a) said first FRT, (b) at least one restrictionendonuclease recognition site, (c) at least one marker gene, (d) abacterial origin of replication, and optionally (e) a mammalian cellularor vital origin of DNA replication.
 14. A DNA construct comprising, asan autonomous fragment, in the following order, reading from 5' to 3'along said fragment:(a) a first FLP recombination target site, (b) aninsert segment comprising:(1) at least one restriction endonucleaserecognition site, (2) at least one marker gene, (3) a bacterial originof replication, and optionally (4) a mammalian cellular or viral originof DNA replication, and (c) a second FLP recombination target site intandem with said first FLP recombination target site.
 15. A method forthe assembly of functional gene(s) for expression in mammalian cells, byrecombination of individual inactive gene segments from one or moregene(s) of interest, wherein each of said segments contains at least oneFRT, said method comprising:contacting said individual inactive genesegments with a FLP recombinase, under conditions suitable forrecombination to occur, thereby providing upon recombination a DNA whichencodes a functional gene of interest, the expression product of whichis biologically active.
 16. A method according to claim 15 wherein saidFLP recombinase is from a species of the genus Saccharomyces.
 17. Amethod according to claim 15 wherein said FLP recombinase is from astrain of Saccharomyces cerevisiae.
 18. A method according to claim 17wherein said FLP recombinase is encoded by the sequence set forth as SEQID NO:1.
 19. An isolated mammalian cell, Wherein the genomic DNA of saidcell contains an inserted heterologous first DNA comprising at least oneFLP recombination target site therein.
 20. An isolated mammalian cellaccording to claim 19 wherein said first DNA comprises at least a firstsegment of one or more first gene(s) of interest.
 21. An isolatedmammalian cell according to claim 20, further comprising DNA encoding,and capable of expressing, in said cell, a FLP recombinase.
 22. Anisolated mammalian cell according to claim 20 wherein said first gene(s)of interest provide a readily analyzable marker feature upon expression.23. An isolated mammalian cell according to claim 19 wherein said FLPrecombination target site has the sequence:

    5'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3'.


24. An isolated mammalian cell according to claim 20 further comprisinga second DNA, wherein said second DNA comprises:(a) at least a secondsegment of said first gene of interest, or (b) at least a segment of asecond gene of interest;wherein said second DNA contains at least oneFLP recombination target site; and wherein said second DNA, whencombined in reading frame with said first DNA, provides a functionalgene.